For 30 years, an effective vaccine has been sought to prevent genital herpes caused by herpes simplex virus 2 (HSV-2). Most of the research has focused on the development of non-replicating HSV-2 vaccine agents such as the glycoprotein D subunit or replication-defective HSV-2 viruses. Such approaches merit consideration. However, it is unclear that non-replicating HSV-2 vaccines elicit the type of life-long immunity against genital herpes that is sought. For each year that elapses without an effective vaccine, another 10 to 20 million people contract HSV-2 infections. Given the scope of the problem, perhaps it is time to consider a second possibility: a live, replicating HSV-2 vaccine strain may be more effective. Most of our successes in controlling viral disease in the human population have been based upon live, replicating viruses. Live vaccinia virus (the original vaccine) was used to end smallpox epidemics. Poliomyelitis, mumps, measles, rubella, and chickenpox are prevented with childhood vaccines that contain live, replicating viruses that occur in nature, but are attenuated in their disease-causing potential. This, our most successful vaccination modality, has not been adequately considered for its potential to control genital herpes. In large part, this is due to the misconception that a live HSV-2 vaccine strain would be dangerous. The P.I.'s published studies and preliminary data establish that genetic engineering combined with current knowledge of HSV biology may be applied to derive live, replicating HSV-1 and HSV-2 viral vaccines that are safe and immunogenic. In principle, attenuation of HSV is readily achieved because ~30 of 75 HSV genes are not essential for viral replication. Many of these genes, such as the ICP0 gene, are required for HSV to resist repression by the host immune response. The P.I. has worked with interferon-sensitive HSV-1 ICP0- viruses for 10 years. Disruption of the ICP0 gene renders HSV-1 and HSV-2 hypersensitive to repression by interferon-1/2, avirulent in animals, and yet these viruses may serve as powerful immunogens. Mice immunized with a live, replicating HSV-1 ICP0- virus are immune to lethal challenge with 1000 times the LD50 dose of HSV-1 (McKrae strain) or HSV-2 (MS strain). Likewise, mice in which an HSV-2 ICP0- virus replicates at the site of immunization are immune to lethal challenge with HSV-2 MS. A systematic effort has not been made to develop a live and appropriately attenuated HSV-2 virus that may establish an inapparent infection at the site of immunization. The P.I.'s data indicate that HSV-1 or HSV-2's full immunogenic potential is only realized when viral replication occurs in the host. If this hypothesis is correct, then live, replicating HSV vaccine strains may be far more protective than any non-replicating HSV vaccine considered to date. Two years of R21 funding is requested to test this hypothesis, and to begin developing HSV-2 ICP0- viruses that may later be used in human clinical trials if a live HSV-2 vaccine strain proves to be safe and effective in protecting mice, guinea pigs, rabbits, and hamsters against genital herpes. PUBLIC HEALTH RELEVANCE: Interferon-sensitive herpes simplex virus-2 (HSV-2) ICP0- viruses are proposed as a live, replicating HSV-2 vaccine strain. Such live HSV-2 vaccine strains may be capable of preventing the spread of genital herpes, a disease that currently afflicts ~50 million people worldwide. The work proposed herein will test a hypothesis that live, replicating HSV-2 ICP0- viruses provide superior protection against exogenous HSV-2 infections relative to non-replicating HSV-2 vaccines that have been the focus of research for 30 years. It is anticipated that a new live HSV-2 ICP0- vaccine strain will emerge from these studies that warrants advancement to human clinical trials.